The Hermansky-Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky-Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329-332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed approximately 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of approximately 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky-Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.